EP2114446A1 - Vaccine - Google Patents
VaccineInfo
- Publication number
- EP2114446A1 EP2114446A1 EP08707821A EP08707821A EP2114446A1 EP 2114446 A1 EP2114446 A1 EP 2114446A1 EP 08707821 A EP08707821 A EP 08707821A EP 08707821 A EP08707821 A EP 08707821A EP 2114446 A1 EP2114446 A1 EP 2114446A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- saccharide
- vaccine
- immunogenic composition
- protein carrier
- conjugate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/095—Neisseria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0275—Salmonella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
- A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to improved methods of conducting carbodiimide condensation reactions.
- it relates to the conjugation of saccharides and proteins using carbodiimide condensation.
- It also relates to immunogenic compositions that may be made comprising the saccharide-protein conjugates of the invention.
- Conjugates can be prepared by direct reductive amination methods as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.
- the conjugation method may alternatively rely on activation of hydroxyl groups of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.
- CDAP 1-cyano-4-dimethylamino pyridinium tetrafluoroborate
- the activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein.
- the cyanate ester can be coupled with hexane diamine or adipic acid dihydrazide (ADH or AH) and the amino-derivatised saccharide is conjugated to the carrier protein using using carbodiimide (e.g. EDAC or EDC) chemistry via a carboxyl group on the protein carrier.
- carbodiimide e.g. EDAC or EDC
- Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094. See also Chu C. et al Infect. Immunity, 1983 245 256.
- Carboxyl for instance via aspartic acid or glutamic acid
- Carboxyl which may be conjugated to natural or derivatised amino groups on saccharide moieties using carbodiimide chemistry
- B) Amino group (for instance via lysine) which may be conjugated to natural or derivatised carboxyl groups on saccharide moieties using carbodiimide chemistry;
- G) lndolyl group (for instance via tryptophan).
- lndolyl group (for instance via tryptophan).
- groups can be used for a coupling: OH, COOH or NH2.
- Aldehyde groups can be generated after different treatments known in the art such as: periodate, acid hydrolysis, hydrogen peroxide, etc.
- carbodiimide chemistry e.g. using EDAC
- EDAC electrospray oxidation deposition
- the chemical is relatively unstable at its reaction pH (4.5-6.5), and therefore all components of the saccharide/protein/carbodiimide conjugation reaction tend to be added together in the art.
- a method of conjugating a saccharide to a protein carrier using carbodiimide condensation chemistry comprising the steps of:
- the protein carrier comprises both amino and carboxyl groups and the saccharide comprises either amino or carboxyl groups: a) mixing the saccharide and aliquot of carbodiimide required to perform the conjugation, and b) adding the aliquot of protein carrier required over a period of 35 seconds to 6 hours;
- the saccharide comprises both amino and carboxyl groups and the protein carrier comprises either amino or carboxyl groups: a) mixing the protein carrier and aliquot of carbodiimide required to perform the conjugation, and b) adding the aliquot of saccharide required over a period of 35 seconds to 6 hours;
- carbodiimide Any suitable carbodiimide may be used as long as it is capable of conjugating saccharides and proteins in an aqueous medium.
- the carbodiimide may be EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) [also known as EDC] or it may be a carbodiimide other than EDAC. Where EDAC or EDC is mentioned herein in any embodiment, it is envisioned that any carbodiimide may alternatively be used.
- saccharide throughout this specification may indicate polysaccharide or oligosaccharide and includes both. It may indicate lipopolysaccharide (LPS) or lipooliogosaccharide (LOS).
- LPS lipopolysaccharide
- LOS lipooliogosaccharide
- Polysaccharides such as bacterial polysaccharides
- Source strain e.g. of bacteria
- LOS lipooliogosaccharide
- Polysaccharides can be sized in order to reduce viscosity in polysaccharide samples and/or to improve filterability for conjugated products.
- Oligosaccharides have a low number of repeat units (typically 5-30 repeat units) and are typically hydrolysed polysaccharides.
- protein carrier is intended to cover both small peptides and large polypeptides (>10 kDa). Clearly large polypeptides are more likely to contain both reactive amino and carboxyl groups without any modification.
- “native polysaccharide” refers to a saccharide that has not been subjected to a process, the purpose of which is to reduce the size of the saccharide.
- a polysaccharide can become slightly reduced in size during normal purification procedures. Such a saccharide is still native. Only if the polysaccharide has been subjected to sizing techniques would the polysaccharide not be considered native.
- sized by a factor up to x2 means that the saccharide is subject to a process intended to reduce the size of the saccharide but to retain a size more than half the size of the native polysaccharide.
- X3, x4 etc. are to be interpreted in the same way i.e. the saccharide is subject to a process intended to reduce the size of the polysaccharide but to retain a size more than a third, a quarter etc. the size of the native polysaccharide.
- the 35 second to 6 hour time period in step b) of the method for the addition of the full aliquot of the final component can be 50 seconds to 5 hours, 1 minute to 4 hours, 2 minutes to 3 hours, 3 minutes to 2 hours, 4 to 60 minutes, 5 to 50 minutes, 6 to 40 minutes, 7 to 30 minutes or 8 to 20 minutes. It may be 1 minute to 5 hours, 10 minutes to 4 hours, 20 minutes to 3 hours, 30 minutes to 2 hours, 40 to 90 minutes, or 50 to 70 minutes. This time can be adjusted according to the precise saccharide and protein being conjugated.
- the aliquot of the final component e.g. of carbodiimide, saccharide or protein
- the reaction mixture is added at a constant rate during the time period (this is conveniently achieved using a pump operating at a constant rate).
- it may be added in stages over the time period.
- parts of the aliquot should be added throughout the period. For instance at least one quarter of the aliquot may be added over the first half of the period, and at least one quarter of the aliquot over the second half of the period.
- the total amount of the aliquot 'a' measured, for instance, in ml. or mg may be added in 4-100 stages ('s') throughout the period.
- the stages are arranged such that an even amount (a/s) is introduced at all the stages.
- the stages are evenly spaced throughout the period 'p' (in seconds). Thus if one stage takes place at time zero of the period 'p', then each subsequent stage could take place at a time which is p/(s-1 ).
- the volume of the aliquot of the final component added in step b) may be adjusted in terms of ease of addition of the aliquot to the reaction within the desired time period.
- the carbodiimide may be added as an aqueous solution (typically buffered at pH 7.5 before being added to the reaction) or as solid powder (EDAC for instance is highly soluble in aqueous media).
- a slow dissolving carbodiimide may be used such that the entire aliquot of powder is added to the reaction all at once but it dissolves at a rate consistent with the desired period over which the aliquot is to be made available to the reaction.
- the protein and/or saccharide may be derivatised to give it one (or to give it the other it does not already have).
- a saccharide only comprising reactive hydroxyl groups e.g. meningococcal serogroup A capsular saccharide
- such a group should be used for derivatising on amino or carboxyl groups so that EDAC condensation may be carried out. This may take place within a repeat subunit, or may be a group only present at the end of the saccharide molecule.
- a saccharide or protein already has amino or carboxyl groups only (e.g. Vi saccharide from Salmonella typhi which naturally has carboxyl but not amino groups), derivatisation can take place to give it the other type of group (i.e. amino groups for Vi). It should be noted, however, that as derivatisation can be partial this action can change the preferred reaction of the invention from a type I to a type III. For instance if Vi saccharide is conjugated to a protein carrier comprising both amino and carboxyl groups situation I adds the aliquot of protein slowly in step b). If the Vi saccharide carboxyl group is partially derivatised with amino groups it will have both carboxyl and amino groups, thus situation III adding the aliquot of carbodiimide slowly in step b) becomes most relevant.
- Derivatisation may occur through the addition of a hetero- or homo-bifunctional linker. It may take place with similar chemistry as described above for saccharide-protein conjugation step (e.g. CDAP or carbodiimide chemistry).
- the linker may have between 4 and 20, 4 and 12, or 5 and 10 carbon atoms. It may have two reactive amino groups, two reactive carboxyl groups, or one of each (e.g. hexane diamine, 6-aminocaproic acid, or adipic acid dihydrazide).
- derivatization takes place through reacting a large excess of the linker with the saccharide and/or protein carrier to be derivatised.
- the saccharide comprises a reactive hydroxyl group as part of its repeating unit which is partially derivatised via an amino group on the linker (e.g. with CDAP chemistry).
- the saccharide comprises a reactive amino group as part of its repeating unit which is partially derivatised via a carboxyl group on the linker (e.g. with carbodiimide chemistry).
- the saccharide comprises a reactive carboxyl group as part of its repeating unit which is partially derivatised via an amino group on the linker (e.g.
- carbodiimide chemistry for instance wherein the carbodiimide in the partial derivatisation step is present at 0.01-0.5, 0.015-0.1 , 0.02-0.075, or 0.025-0.05 mg carbodiimide / mg saccharide).
- the aliquot of carbodiimide required to perform the conjugation is 0.01 to 3, 0.05 to 2, or 0.09 to 1 mg carbodiimide/mg saccharide (for instance 0.07 to 0.25, or 0.1 to 0.2 mg/mg saccharide).
- these numbers are calculated in respect of EDAC being the carbodiimide, these numbers may optionally be adjusted if any other carbodiimide is used by multiplying the numbers in the range by: (molecular weight of other carbodiimide)/(molecular weight of EDAC).
- the saccharide may be present in the methods of the invention at a final concentration of 0.5-50 mg/ml in step b). This will depend on the size and nature of the saccharide, and the extent of any derivatisation. For instance for oligosaccharides a larger concentration will be required, but for large polysaccharides a much smaller concentration will be more appropriate. If it is towards the high end of partially derivatised with amino or carboxyl groups a smaller concentration may be appropriate to reduce the possibility of any cross-linking.
- the protein carrier may be present at a final concentration of 1-50 mg/ml in step b).
- the initial ratio of protein carrier to saccharide in the methods of the invention can be 5:1 to 1 :5, 4:1 to 1 :1 , or 3:1 to 2:1 (w/w). Again this will depend on the size and nature of the saccharide, and the extent of any derivatisation.
- Salt conditions may also be varied according to the nature of the saccharide/protein. Usually around 0.2M NaCI may be present in step b) of the methods of the invention, but may be 0-2, 0.1-1 or 0.2-0.5 M.
- the reaction pH may be any pH where the carbodiimide is activated - for instance pH 4.5-6.5, 4.7-6.0, or 5-5.5. This pH is typically maintained throughout the reaction by addition of acid/base as required. EDAC is usually stable at pH 7.5, though if the conjugation requires to be done at higher pH compounds which are known to keep the reaction intermediate stable (such as N- hydroxysuccinimide) may also be present in the reaction in step b), in which case the reaction pH in step b) may be maintained at pH 4.5-7.5.
- the reaction temperature during step b) of the methods of the invention can be 4-37, 10- 32, 17-30, or 22-27 0 C, and is typically maintained throughout the reaction.
- step b) the reaction is typically maintained for a further 10 minutes to 72 hours, 20 minutes to 48 hours, 30 minutes to 24 hours, 40 minutes to 12 hours, 50 minutes to 6 hours, or 1-3 hours, for instance 10-120, 10-80, 10-50, 20-40, or 25-30 minutes.
- the pH is adjusted to 7.5-9 (towards the higher end of this if N- hydroxysuccinimide is present) to go back to the stable pH range of carbodiimide.
- the saccharide-protein conjugate may be purified from: unreacted components, free saccharide, etc by injecting it on a size exclusion chromatography column (for instance Sephacryl S400HR, Pharmacia). This is typically carried out at 2-8 0 C.
- the conjugate may be sterile filtered then stored.
- an effective dose for instance 1-20, 2-15, or 3-10 ⁇ g saccharide /dose
- a pharmaceutically acceptable excipient for instance a salt or adjuvant
- any saccharide of viral, fungal, bacterial or eukaryotic source may be conjugated using the methods of the invention. It may be the Vi saccharide from Salmonella typhi, or a saccharide other than Vi. It may be the capsular saccharide Hib from H. influenzae type b, or may be a saccharide other than Hib. In one embodiment the saccharide is a bacterial capsular saccharide, for instance derived from a bacterium selected from a list consisting of: N.
- MenA meningitidis serogroup A
- MenB meningitidis serogroup B
- C meningitidis serogroup C
- W135 MenW
- Y MenY
- Group B Streptococcus group Ia, Ib, II, III, IV, V, Vl, or VII Staphylococcus aureus type 5, Staphylococcus aureus type 8, Salmonella typhi (Vi saccharide), Vibrio cholerae, or H. influenzae type b.
- the weight-average molecular weight of the saccharide may be 1000-2000000, 5000- 1000000, 10000-500000, 50000-400000, 75000-300000, or 100000-200000.
- the molecular weight or average molecular weight of a saccharide herein refers to the weight- average molecular weight (Mw) of the saccharide measured prior to conjugation and is measured by MALLS.
- Mw weight- average molecular weight
- the MALLS technique is well known in the art and is typically carried out as described in example 2.
- two columns (TSKG6000 and 5000PWxI) may be used in combination and the saccharides are eluted in water.
- Saccharides are detected using a light scattering detector (for instance Wyatt Dawn DSP equipped with a 1 OmW argon laser at 488nm) and an inferometric refractometer (for instance Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498nm).
- n an embodiment, the polydispersity of the saccharide is 1-1.5, 1-1.3, 1-1.2, 1- 1.1 or 1-1.05 and after conjugation to a carrier protein, the polydispersity of the conjugate is 1.0-2.5, 1.0-2.0. 1.0-1.5, 1.0-1.2, 1.5-2.5, 1.7-2.2 or 1.5-2.0. All polydispersity measurements are by MALLS.
- the saccharide may be either a native polysaccharide or may have been sized by a factor of no more than 2, 4, 6, 8, 10 or 20 fold (for instance by microfluidization [e.g. by Emulsiflex C-50 apparatus] or other known technique [for instance heat, chemical, oxidation, sonication methods]). Oligosaccharides may have been sized substantially further [for instance by known heat, chemical, or oxidation methods].
- the saccharide may be a bacterial lipooligosaccharide or lipopolysaccharide (see above table), for instance derived from a bacterium selected from a list consisting of: N. meningitidis, H. influenzae, E. coli, Salmonella or M. catarrhalis.
- the LOS may be meningococcal immunotype L2, L3 or L10. It may be detoxified by alkaline treatment of its Lipid A moiety.
- the MenA capsular saccharide is at least partially O-acetylated such that at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at at least one position. O-acetylation is for example present at least at the 0-3 position of at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.
- the MenC capsular saccharide is is at least partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of ( ⁇ 2 ⁇ 9)-linked NeuNAc repeat units are O-acetylated at at least one or two positions.
- O-acetylation is for example present at the 0-7 and/or 0-8 position of at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.
- the MenW capsular saccharide is at least partially O-acetylated such that at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at at least one or two positions.
- O-acetylation is for example present at the 0-7 and/or 0-9 position of at least 30%. 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.
- the MenY capsular saccharide is at least partially O-acetylated such that at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units are O-acetylated at at least one or two positions.
- O-acetylation is present at the 7 and/or 9 position of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98% of the repeat units.
- the percentage of O- acetylation refers to the percentage of the repeat units containing O-acetylation. This may be measured in the saccharide prior to conjugate and/or after conjugation.
- the protein carrier may be any peptide or protein. It may comprise one or more T-helper epitopes.
- the protein carrier is selected from the group consisting of: TT, DT, CRM197, fragment C of TT, protein D of H. influenzae, pneumococcal PhtD, and pneumococcal Pneumolysin.
- the carrier protein may be tetanus toxoid (TT), tetanus toxoid fragment C, non-toxic mutants of tetanus toxin [note all such variants of TT are considered to be the same type of carrier protein for the purposes of this invention], diphtheria toxoid (DT), CRM197, other non-toxic mutants of diphtheria toxin [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida et al J. Biol. Chem.
- meningitidis serogroup B - EP0372501 meningitidis serogroup B - EP0372501
- synthetic peptides EP0378881 , EP0427347
- heat shock proteins WO 93/17712, WO 94/03208
- pertussis proteins WO 98/58668, EP0471 177
- cytokines lymphokines, growth factors or hormones
- artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens (Falugi et al (2001 ) Eur J Immunol 31 ; 3816-3824) such as N19 protein (Baraldoi et al (2004) Infect lmmun 72; 4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptake proteins (WO 01/72337), toxin A or B of C.
- H. influenzae Protein D (EP594610 and WO 00/56360), pneumococcal PhtA (WO 98/18930, also referred to Sp36), pneumococcal PhtD (disclosed in WO 00/37105, and is also referred to SpO36D), pneumococcal PhtB (disclosed in WO 00/37105, and is also referred to SpO36B), or PhtE (disclosed in WO00/30299 and is referred to as BVH-3).
- a saccharide-protein carrier conjugate (or an immunogenic composition or vaccine) obtainable or obtained by the method of the invention.
- the methods of the invention may be incorporated within a method of making an immunogenic composition or vaccine of the invention through carrying out the conjugation method of the invention and formulating the resulting saccharide-protein carrier conjugate in an immunogenic composition or vaccine (for example by formulating the conjugate with a pharmaceutically acceptable excipient).
- a use of the immunogenic composition or vaccine of the invention in the manufacture of a medicament for the prevention or treatment of disease, and a method of preventing or treating disease comprising the step of administering an effective dose of the immunogenic composition or vaccine of the invention to a patient in need thereof is further provided.
- the use or method may be such that the disease is caused by a bacterium selected from a list consisting of: N. meningitidis, Streptococcus pneumoniae, M. catarrhalis, Group B Streptococcus, Staphylococcus aureus, Salmonella typhi, Vibrio cholerae, E. coli, and H. influenzae.
- the immunogenic compositions of the invention may also comprise a DTPa or DTPw vaccine (for instance one containing DT, TT, and either a whole cell pertussis (Pw) vaccine or an acellular pertussis (Pa) vaccine (comprising for instance pertussis toxoid, FHA, pertactin, and, optionally agglutinogens 2 and 3).
- a DTPa or DTPw vaccine for instance one containing DT, TT, and either a whole cell pertussis (Pw) vaccine or an acellular pertussis (Pa) vaccine (comprising for instance pertussis toxoid, FHA, pertactin, and, optionally agglutinogens 2 and 3).
- Pw whole cell pertussis
- Pa acellular pertussis
- Such combinations may also comprise a vaccine against hepatitis B (for instance it may comprise hepatitis B surface antigen [HepB], optionally ads
- the immunogenic composition of the invention comprises Hib, MenA and MenC saccharide conjugates, or Hib and MenC saccharide conjugates, or Hib, MenC and MenY saccharide conjugates, or MenA, MenC, MenW and MenY saccharide conjugates, wherein at least one, two or all the saccharide conjugates are made according the method of the invention.
- Immunogenic compositions of the invention optionally comprise additional viral antigens conferring protection against disease caused by measles and/or mumps and/or rubella and/or varicella.
- immunogenic composition of the invention contains antigens from measles, mumps and rubella (MMR) or measles, mumps, rubella and varicella (MMRV).
- MMR measles, mumps and rubella
- MMRV measles, mumps, rubella and varicella
- these viral antigens are optionally present in the same container as the meningococcal and/or Hib saccharide conjugate(s) present in the composition.
- these viral antigens are lyophilised.
- the immunogenic composition of the invention further comprises an antigen from N. meningitidis serogroup B.
- the antigen is optionally an outer membrane vesicle preparation from N. meningitidis serogroup B as described in EP301992, WO 01/09350, WO 04/14417, WO 04/14418 and WO 04/14419.
- the immunogenic composition of the invention may comprise a dose of each saccharide conjugate between 0.1 and 20 ⁇ g, 2 and 10 ⁇ g, 2 and 6 ⁇ g or 4 and 7 ⁇ g of saccharide.
- the immunogenic composition of the invention is adjusted to or buffered at, or adjusted to between pH 7.0 and 8.0, pH 7.2 and 7.6 or around or exactly pH 7.4.
- the immunogenic composition or vaccines of the invention are optionally lyophilised in the presence of a stabilising agent for example a polyol such as sucrose or trehalose.
- a stabilising agent for example a polyol such as sucrose or trehalose.
- the immunogenic composition or vaccine of the invention contains an amount of an adjuvant sufficient to enhance the immune response to the immunogen.
- Suitable adjuvants include, but are not limited to, aluminium salts (aluminium phosphate or aluminium hydroxide), squalene mixtures (SAF-1 ), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875.
- ICOMs immunostimulating complexes
- the immunologically effective amounts of the immunogens must be determined empirically. Factors to be considered include the immunogenicity, whether or not the immunogen will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, route of administrations and the number of immunising dosages to be administered.
- the active agent can be present in varying concentrations in the pharmaceutical composition or vaccine of the invention.
- the minimum concentration of the substance is an amount necessary to achieve its intended use, while the maximum concentration is the maximum amount that will remain in solution or homogeneously suspended within the initial mixture.
- the minimum amount of a therapeutic agent is optionally one which will provide a single therapeutically effective dosage.
- the minimum concentration is an amount necessary for bioactivity upon reconstitution and the maximum concentration is at the point at which a homogeneous suspension cannot be maintained.
- the amount is that of a single therapeutic application.
- each dose will comprise 1-100 ⁇ g of protein antigen, optionally 5-50 ⁇ g or 5-25 ⁇ g.
- doses of bacterial saccharides are 10-20 ⁇ g, 5-1 O ⁇ g, 2.5-5 ⁇ g or 1-2.5 ⁇ g of saccharide in the conjugate.
- the vaccine preparations of the present invention may be used to protect or treat a mammal (for example a human patient) susceptible to infection, by means of administering said vaccine via systemic or mucosal route.
- a human patient is optionally an infant (under 12 months), a toddler (12-24, 12-16 or 12-14 months), a child (2-10, 3-8 or 3-5 years) an adolescent (12-21 , 14-20 or 15-19 years) or an adult.
- These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts.
- Intranasal administration of vaccines for the treatment of pneumonia or otitis media is preferred (as nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at its earliest stage).
- the vaccine of the invention may be administered as a single dose, components thereof may also be co-administered together at the same time or at different times (for instance if saccharides are present in a vaccine these could be administered separately at the same time or 1-2 weeks after the administration of a bacterial protein vaccine for optimal coordination of the immune responses with respect to each other).
- 2 different routes of administration may be used.
- viral antigens may be administered ID (intradermal), whilst bacterial proteins may be administered IM (intramuscular) or IN (intranasal). If saccharides are present, they may be administered IM (or ID) and bacterial proteins may be administered IN (or ID).
- the vaccines of the invention may be administered IM for priming doses and IN for booster doses.
- Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach” (eds Powell M. F. & Newman M.J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877.
- a further aspect of the invention is a process for making the immunogenic composition or vaccine of the invention, comprising the step of mixing the MenA and MenC saccharides of the invention made by the method of the invention, with MenW and MenY that have not been made according to the invention, and with a pharmaceutically acceptable excipient.
- Salmonella typhi immunogenic compositions/vaccines of the invention Salmonella typhi immunogenic compositions/vaccines of the invention
- the immunogenic composition or vaccine of the invention comprises a Vi saccharide-protein carrier conjugate made according to the processes of the invention and a pharmaceutically acceptable excipient.
- the Vi saccharide-protein carrier conjugate may comprise 0.5-15, 1-10, 2.0-7.5 or 2.5-5 ⁇ g of Vi saccharide per human dose.
- the Vi saccharide from Salmonella typhi in the conjugate may be the same as that in the registered product Typherix® (GlaxoSmithKline Biologicals s.a.), described in EP1 107787.
- the Vi saccharide conjugates of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide, or aluminium phosphate, or a mixture of both aluminium hydroxide and aluminium phosphate.
- the Vi saccharide conjugate may be unadsorbed onto an adjuvant, e.g. an aluminium adjuvant salt.
- the immunogenic composition or vaccine of the invention further comprises a Hib capsular saccharide-protein carrier conjugate.
- a Hib capsular saccharide-protein carrier conjugate This may be made according to the process of the invention, or by any method known in the art.
- it may be the Hiberix® product of GlaxoSmithKline Biologicals s.a.
- the covalent binding of Haemophilus influenzae (Hib) PRP polysaccharide to TT may be carried out by the coupling chemistry developed by Chu et al (Infection and Immunity 1983, 40 (1 ); 245- 256).
- Hib PRP polysaccharide is activated by adding CNBr and incubating at pH10.5 for 6 minutes.
- the pH is lowered to pH8.75 and adipic acid dihydrazide (ADH) is added and incubation continued for a further 90 minutes.
- the activated PRP may be coupled to, for instance, purified tetanus toxoid via carbodiimide condensation using 1-ethyl-3-(3- dimethyl-aminopropyl)carbodiimide (EDAC).
- EDAC is added to the activated PRP to reach a final ratio of 0.6mg EDAC/mg activated PRP.
- the pH is adjusted to 5.0 and purified tetanus toxoid added to reach 2mg TT/mg activated PRP.
- the resulting solution is left for three days with mild stirring.
- the conjugate may be purifed on a sephacryl S500HR (Pharmacia, Sweden) column equilibrated in 0.2M NaCI.
- Hib antigen conjugate may optionally be adsorbed onto aluminium phosphate as described in WO97/00697, or may be unadsorbed as described in WO02/00249 or may not have undergone a specific process for adsorption.
- an antigen being 'unadsorbed onto an aluminium adjuvant salt' herein it is meant that an express or dedicated adsorption step for the antigen on fresh aluminium adjuvant salt is not involved in the process of formulating the composition.
- Hib is present at a low dose (e.g. 1-6 ⁇ g, 2-4 ⁇ g or around or exactly 2.5 ⁇ g of saccharide) as described in WO 02/00249.
- the Hib saccharide conjugate is present in a lower saccharide dose than the saccharide dose of the Vi saccharide conjugate.
- the Hib saccharide conjugate may comprise 0.1-9, 1-5, or 2-3 ⁇ g of saccharide per human dose (normally 0.5 ml_).
- the Hib saccharide may be conjugated to any protein carrier described herein, for example one selected from the group consisting of TT, DT, CRM197, fragment C of TT, protein D, OMPC and pneumolysin.
- the same protein carrier is used (e.g. independently) in the Hib saccharide conjugate and the Vi saccharide conjugate, for instance TT.
- the ratio of Hib saccharide to protein carrier in the Hib saccharide conjugate may be between 1 :5 and 5:1 (w/w), for instance between 1 :1 and 1 :4, 1 :2 and 1 :3.5 or around 1 :3 (w/w).
- the Hib saccharide may be conjugated to the protein carrier via a linker, which is typically bifunctional (homo or hetero bifunctional).
- the linker may have two reactive amino groups (one on each end), or two reactive carboxylic acid groups, or a reactive amino group at one end and a reactive carboxylic acid group at the other end.
- the linker may have between 4 and 12 carbon atoms. In one aspect the linker is ADH.
- the Hib saccharide may be conjugated to the protein carrier or linker using CNBr or CDAP.
- the protein carrier may be conjugated to the Hib saccharide or linker using carbodiimide chemistry, optionally EDAC chemistry.
- Further vaccine combinations involving the Vi and/or Hib conjugate antigen in which the Vi conjugates of the present invention may be used are described in PCT/EP2006/006210, PCT/EP2006/006188, PCT/EP2006/006269, PCT/EP2006/006268, or PCT/EP2006/006220.
- Vi or Vi + Hib conjugates in the immunogenic compositions or vaccines of the invention are mixed with further antigens.
- a DTP (DTPa or DTPw) vaccine a Hepatitis B vaccine/antigen such as hepatitis B surface antigen, optionally adsorbed onto aluminium phosphate
- a Hepatitis A vaccine/antigen such as an inactivated hepatitis A virus preparation
- a Polio virus vaccine/antigen such as an inactivated polio virus (IPV) preparation (optionally comprising types 1 , 2 and 3)
- one or more meningococcal capsular saccharide - protein carrier conjugates [where the capsular saccharide(s) are derived from the following meningococcal serogroups: A, C, W135, Y, A and C, A and W135, A and Y, C and W135, C and Y, W
- Vi and Hib capsular saccharide conjugates are co-lyophilised, optionally in the presence of a stabilising agent for example a polyol such as sucrose and/or trehalose.
- the lyophilised formulation may further comprise one or more meningococcal capsular saccharide - protein carrier conjugates (where the capsular saccharide(s) are derived from the following meningococcal serogroups: A, C, W135, Y, A and C, A and W135, A and Y, C and W135, C and Y, W135 and Y, A and C and W135, A and C and Y, A and W135 and Y, C and W135 and Y, A and C and W135 and Y, A and C and W135 and Y).
- the lyophilised composition of the invention may be reconstituted with an aqueous medium prior to administration.
- the aqueous medium may be buffered. It may have further antigens for instance those listed above not already included in the lyophilised composition [e.g.
- a DTP (DTPa or DTPw) vaccine a Hepatitis B vaccine/antigen such as hepatitis B surface antigen, optionally adsorbed onto aluminium phosphate
- a Hepatitis A vaccine/antigen such as an inactivated hepatitis A virus preparation
- a Polio virus vaccine/antigen such as an inactivated polio virus (IPV) preparation (optionally comprising types 1 , 2 and 3)
- one or more meningococcal capsular saccharide - protein carrier conjugates [where the capsular saccharide(s) are derived from the following meningococcal serogroups: A, C, W135, Y, A and C, A and W135, A and Y, C and W135, C and Y, W135 and Y, A and C and W135, A and C and Y, A and W135 and Y, A and W135 and Y, A and W135 and Y, A and W135 and and
- the immunogenic composition or vaccines of the invention may contain aluminium phosphate, aluminium hydroxide or a mixture of both. Alternatively it may contain no aluminium salts, or may be unadjuvanted.
- the immunogenic composition or vaccine of the invention may be buffered at between pH 7.0 and 8.0.
- a vaccine kit for concomitant or sequential administration comprising two multi-valent immunogenic compositions for conferring protection in a host against disease caused by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Salmonella typhi and Haemophilus influenzae, said kit comprising a first container comprising :
- TT tetanus toxoid
- DT diphtheria toxoid
- Pa whole cell or acellular pertussis components
- the first container may further comprise hepatitis B surface antigen, optionally adsorbed on aluminium phosphate.
- the first or second container may further comprise inactivated polio virus (IPV).
- IPV inactivated polio virus
- a use of the immunogenic composition or vaccine or kit of the invention in the manufacture of a medicament for the prevention or treatment of disease is also provided, as is method of preventing or treating disease comprising the step of administering an effective dose of the immunogenic composition or vaccine of the invention to a patient in need thereof.
- the use or method of the invention may be in respect of diseases caused by one or more bacteria selected from a list consisting of: N. meningitidis, Salmonella typhi, H. influenzae, Bordetella pertussis, Clostridium tetani, and Corynebacterium diphtheriae.
- DTP vaccines are well known vaccines to prevent or treat diphtheria, tetanus and B. pertussis disease.
- the vaccines of the invention may comprise diphtheria, tetanus and/or pertussis component(s).
- the diphtheria antigen is typically a diphtheria toxoid.
- the preparation of diphtheria toxoids (DT) is well documented. Any suitable diphtheria toxoid may be used.
- DT may be produced by purification of the toxin from a culture of Corynebacterium diphtheriae followed by chemical detoxification, but is alternatively made by purification of a recombinant, or genetically detoxified analogue of the toxin (for example, CRM197, or other mutants as described in US 4,709,017, US 5,843,71 1 , US 5,601 ,827, and US 5,917,017).
- the diphtheria toxoid of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide.
- the diphtheria toxoid of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate.
- the diphtheria toxoid may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate.
- the tetanus antigen of the invention is typically a tetanus toxoid.
- Methods of preparing tetanus toxoids (TT) are well known in the art.
- TT is produced by purification of the toxin from a culture of Clostridium tetani followed by chemical detoxification, but is alternatively made by purification of a recombinant, or genetically detoxified analogue of the toxin (for example, as described in EP 209281 ). Any suitable tetanus toxoid may be used.
- Tetanus toxoid' may encompass immunogenic fragments of the full-length protein (for instance Fragment C - see EP 478602).
- the tetanus toxoid of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide.
- the tetanus toxoid of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate.
- the tetanus toxoid may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate.
- the pertussis component of the invention may be either acellular (Pa) where purified pertussis antigens are used or whole-cell (Pw) where killed whole cell pertussis is used as the pertussis component.
- Pw may be inactivated by several methods, including mercury free methods. Such methods may include heat (e.g. 55-65°C or 56-60 0 C, for 5-60 minutes or 10-30 minutes, e.g. 60 0 C for 30 minutes), formaldehyde (e.g. 0.1 % at 37°, 24 hours), glutaraldehyde (e.g. 0.05% at room temperature, 10 minutes), acetone-l (e.g. three treatments at room temperature) and acetone-l I (e.g. three treatments at room temperature and fourth treatment at 37°C) inactivation (see for example Gupta et al.,
- Thiomersal has been used in the past in the preparation of killed whole-cell Bordetella pertussis. However, in one embodiment it is not used in the formulation process of the vaccines of the present invention.
- a Pw dose of 5-50 lOU, 7-40 lOU, 9-35 lOU, 11-30 lOU, 13-25 lOU, 15-21 IOU or around or exactly 20 IOU is typically used.
- Acellular Pa vaccines are also well known, and may comprise 2 or more antigens from: pertussis toxoid [or known detoxified genetic mutants of pertussis toxin] (PT), filamentous haemagglutinin (FHA), pertactin (PRN), agglutinogens 2 & 3.
- the Pa vaccine comprises PT, FHA and PRN.
- the pertussis component of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide. In another embodiment, the pertussis component of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate. In a further embodiment the pertussis component may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate.
- Hepatitis B antigen/vaccine The preparation of Hepatitis B surface antigen (HBsAg) is well documented. See for example, Hartford et al., 1983, Develop. Biol. Standard 54:125, Gregg et al., 1987, Biotechnology 5:479, EP0226846, EP0299108. It may be prepared as follows. One method involves purifying the antigen in particulate form from the plasma of chronic hepatitis B carriers, as large quantities of HBsAg are synthesised in the liver and released into the blood stream during an HBV infection. Another method involves expressing the protein by recombinant DNA methods.
- the HBsAg may be prepared by expression in the Saccharomyces cerevisiae yeast, pichia, insect cells (e.g. Hi5) or mammalian cells.
- the HBsAg may be inserted into a plasmid, and its expression from the plasmid may be controlled by a promoter such as the "GAPDH" promoter (from the glyceraldehyde-3- phosphate dehydrogenase gene).
- the yeast may be cultured in a synthetic medium.
- HBsAg can then be purified by a process involving steps such as precipitation, ion exchange chromatography, and ultrafiltration. After purification, HBsAg may be subjected to dialysis (e.g. with cysteine).
- the HBsAg may be used in a particulate form.
- Hepatitis B surface antigen or "HBsAg” includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg S antigen (see Tiollais et ai, 1985, Nature 317:489 and references therein) HBsAg as herein described may, if desired, contain all or part of a pre-S sequence as described in the above references and in EP0278940.
- the HBsAg may comprise a polypeptide comprising an amino acid sequence comprising residues 133-145 followed by residues 175-400 of the L-protein of HBsAg relative to the open reading frame on a Hepatitis B virus of ad serotype (this polypeptide is referred to as L * ; see EP0414374).
- HBsAg within the scope of the invention may also include the preS1-preS2 -S polypeptide described in EP0198474 (Endotronics) or analogues thereof such as those described in EP0304578 (McCormick and Jones)
- HBsAg as herein described can also refer to mutants, for example the "escape mutant" described in WO 91/14703 or EP051 1855A1 , especially HBsAg wherein the amino acid substitution at position 145 is to arginine from glycine.
- the HBsAg may be in particle form.
- the particles may comprise for example S protein alone or may be composite particles, for example L * , S) where L * is as defined above and S denotes the S-protein of HBsAg.
- L * is as defined above and S denotes the S-protein of HBsAg.
- the said particle is advantageously in the form in which it is expressed in yeast.
- HBsAg is the antigen used in EngerixBTM (GlaxoSmithKline Biologicals S.A.), which is further described in WO93/24148.
- Hepatitis B surface antigen may be adsorbed onto aluminium phosphate, which may be done before mixing with the other components (described in WO93/24148).
- the Hepatitis B component should be substantially thiomersal free (method of preparation of HBsAg without thiomersal has been previously published in EP1307473).
- the vaccines/compositions of the invention may further comprise a capsular saccharide of a bacterium selected from the group consisting of N. meningitidis type A (MenA, optionally conjugated to a carrier protein), N. meningitidis type C (MenC, optionally conjugated to a carrier protein), N. meningitidis type W (MenW, optionally conjugated to a carrier protein), and N. meningitidis type Y (MenY, optionally conjugated to a carrier protein).
- the vaccines of the invention may comprise one or more antigens from the different strains of N.
- meningitidis which may be used alone or in any combination of two, three or four components as detailed below: MenA, MenC, MenW, MenY, or MenA + MenC, MenA + MenW, MenA + MenY, MenC + MenW, MenC + MenY, MenW + MenY or MenA + MenC + MenW, MenA + MenC + MenY, MenA + MenW + MenY, MenC + MenW + MenY or MenA + MenC + MenW + MenY.
- the Neisseria meningitidis component(s) of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide.
- the Neisseria meningitidis component(s) of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate.
- the Neisseria meningitidis component(s) may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate.
- the Neisseria meningitidis component(s) may be unadsorbed onto an adjuvant, e.g. an aluminium adjuvant salt.
- the conjugates may be made by any means - in one embodiment the methods described in PCT/EP2006/006210, PCT/EP2006/006188, PCT/EP2006/006269, PCT/EP2006/006268, or PCT/EP2006/006220 are utilised.
- the conjugates may be made using the conjugation process of the present invention.
- the vaccines of the invention may also comprise a MenB component such as an outer membrane vesicle or bleb as described in WO01/09350, WO03/105890,
- the MenB component(s) of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide.
- the MenB component(s) of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate.
- the MenB component(s) may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate.
- the MenB component(s) may be unadsorbed onto an adjuvant, e.g. an aluminium adjuvant salt.
- Hepatitis A antigen(s)/vaccines A component affording protection against Hepatitis A may be the product known as HavrixTM (Registered Trade Mark of GlaxoSmithKline Biologicals S.A.) which is a killed attenuated vaccine derived from the HM-175 strain of Hepatitis A virus (HAV) (see "Inactivated Candidate Vaccines for Hepatitis A" by F. E. Andre et ai, 1980, Prog. Med. Virol. 37:72 and the product monograph "Havrix” published by SmithKline Beecham Biologicals 1991 ). Flehmig et al. (1990, Prog. Med Virol.
- HAV antigen or "HAV vaccine” or "Hepatitis A vaccine” refers to any antigen capable of stimulating neutralising antibody to HAV in humans.
- the HAV antigen comprises inactivated attenuated virus particles, or in another embodiment it may be a HAV capsid or HAV viral protein, which may conveniently be obtained by recombinant DNA technology.
- the Hepatitis A component of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide.
- the Hepatitis A component of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate.
- the Hepatitis A component may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate.
- the compositions of the invention comprising a Hepatitis A vaccine do not comprise phenol.
- the vaccines of the invention may further comprise Malarial antigen(s).
- the Malarial antigen may be RTS 1 S (hybrid protein between CS and HBsAg - described in US 6,306,625 and EP 0614465).
- RTS, S may be used in the vaccines of the invention in place of HBsAg.
- Other Malarial antigens may also be used in the vaccines of the invention, including CS protein, RTS, TRAP, 16kD protein of B 2992, AMA-1 , MSP1 , optionally including CpG (WO2006/029887, WO98/05355, WO01/00231 ).
- the Malarial antigen(s) of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide.
- the Malarial antigen(s) of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate.
- the Malarial antigen(s) may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate.
- the Malarial antigen is adjuvanted with an oil-in-water emulsion and/or lipid A derivative (such as MPL) and or sterol (such as cholesterol) and/or tocol (such as ⁇ -tocopherol)
- an adjuvant e.g. an aluminium adjuvant salt. Polio Virus antigen(s)/vaccines
- the vaccines of the invention may further comprise antigens affording protection against polio virus.
- IPV Inactivated Polio Virus
- Vaccines/compositions of the invention may include IPV type 1 (e.g. Mahoney or Brunhilde) or IPV type 2 (e.g. MEF-1 ) or IPV type 3 (e.g. Saukett), or IPV types 1 and 2, or IPV types 1 and 3, or IPV types 2 and 3, or IPV types 1 , 2 and 3.
- IPV inactivated poliovirus
- IPV should comprise types 1 , 2 and 3 as is common in the vaccine art, and may be the SaIk polio vaccine which is inactivated with formaldehyde (see for example, Sutter et al., 2000, Pediatr. Clin. North Am. 47:287; Zimmerman & Spann 1999, Am Fam Physician 59:1 13; SaIk et al., 1954, Official Monthly Publication of the American Public Health Association 44(5):563; Hennesen, 1981 , Develop. Biol. Standard 47:139; Budowsky, 1991 , Adv. Virus Res. 39:255).
- the IPV is not adsorbed (e.g. before mixing with other components).
- the IPV component(s) of the invention may be adsorbed onto an aluminium salt such as aluminium hydroxide (e.g. before or after mixing with other components).
- the IPV component(s) of the invention may be adsorbed onto an aluminium salt such as aluminium phosphate.
- the IPV component(s) may be adsorbed onto a mixture of both aluminium hydroxide and aluminium phosphate. If adsorbed, one or more IPV components may be adsorbed separately or together as a mixture. IPV may be stabilised by a particular drying process as described in WO2004/039417.
- Poliovirus may be grown in cell culture.
- the cell culture may be a VERO cell line or PMKC, which is a continuous cell line derived from monkey kidney.
- VERO cells can conveniently be cultured microcarriers.
- Culture of the VERO cells before and during viral infection may involve the use of bovine-derived material, such as calf serum, and this material should be obtained from sources which are free from bovine spongiform encephalitis (BSE). Culture may also involve materials such as lactalbumin hydrolysate.
- virions may be purified using techniques such as ultrafiltration, diafiltration, and chromatography. Prior to administration to patients, the viruses must be inactivated, and this can be achieved by treatment with formaldehyde.
- Viruses may be grown, purified and inactivated individually, and then combined to give a bulk mixture for IPV vaccine use or for addition to the other antigens.
- Antigens in vaccines of the invention will be present in "immunologically effective amounts" i.e. the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention of disease.
- Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).
- Standard doses of available polio vaccines contain 40 D antigen units of inactivated poliovirus type 1 , 8 D antigen units of inactivated poliovirus type 2 and 32 D antigen units of inactivated poliovirus type 3 (e.g. Infanrix-IPVTM).
- the vaccines/compositions of the invention may include a pharmaceutically acceptable excipient such as a suitable adjuvant.
- suitable adjuvants include an aluminium salt such as aluminium hydroxide or aluminium phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, or may be cationically or anionically derivatised saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g.
- MPL monophosphoryl lipid A
- 3-O-deacylated MPL [3D-MPL], quil A, Saponin, QS21 , Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, Ml), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), AS-2 (Smith-Kline Beecham, Philadelphia, PA), CpG oligonucleotides, bioadhesives and mucoadhesives, microparticles, liposomes, polyoxyethylene ether formulations, polyoxyethylene ester formulations, muramyl peptides or imidazoquinolone compounds (e.g. imiquamod and its homologues).
- Human immunomodulators suitable for use as adjuvants in the invention include cytokines such as interleukins (e.g. IL-1 , IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc), macrophage colony stimulating factor (M-CSF), tumour necrosis factor (TNF), granulocyte, macrophage colony stimulating factor (GM-CSF) may also be used as adjuvants.
- the adjuvant composition of the formulations induces an immune response predominantly of the TH1 type. High levels of TH 1 -type cytokines (e.g.
- IFN- ⁇ , TNF ⁇ , IL-2 and IL-12 tend to favour the induction of cell mediated immune responses to an administered antigen.
- the level of TH 1 -type cytokines will increase to a greater extent than the level of TH2-type cytokines.
- the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, 1989, Ann. Rev. Immunol. 7:145.
- suitable adjuvant systems which promote a predominantly TH1 response include, derivatives of lipid A (e.g. of reduced toxicity), Monophosphoryl lipid A
- MPL 3-de-O-acylated monophosphoryl lipid A
- 3D- MPL 3-de-O-acylated monophosphoryl lipid A
- An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.
- a particularly potent adjuvant formulation involving QS21 , 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210.
- the vaccine may additionally comprise a saponin, which may be QS21.
- the formulation may also comprise an oil in water emulsion and tocopherol (WO 95/17210).
- Unmethylated CpG containing oligonucleotides (WO 96/02555) are also preferential inducers of a TH 1 response and are suitable for use in the present invention.
- the vaccines of the invention may also comprise combinations of aspects of one or more of the adjuvants identified above.
- AI(OH) 3 / AIPO 4 ratios may be 0/1 15, 23/92, 69/46, 46/69, 92/23 or 115/0.
- certain components of the vaccines of the invention may be not expressly adsorbed onto adjuvant, in particular aluminium salts.
- IPV may be unadsorbed or adsorbed onto AI(OH) 3
- DT may be adsorbed onto AI(OH) 3 or AIPO 4
- TT may be adsorbed onto AI(OH) 3 or AIPO 4
- Pw may be adsorbed onto or mixed with AIPO 4
- PRN may be adsorbed onto AI(OH) 3
- FHA may be adsorbed onto AI(OH) 3
- PT may be adsorbed onto AI(OH) 3
- HB HepB surface antigen
- Hib may be adsorbed onto AIPO 4 or unadsorbed
- Men ACWY may be adsorbed onto AI(OH) 3 or AIPO 4 or unadsorbed
- MenB may be adsorbed onto AI(OH) 3 or AIPO 4 or unadsorbed
- Vi may be adsorbed onto AI(OH) 3 or AIPO 4 or unadsorbed
- HepA may be adsorbed onto AI(OH) 3 Or AIPO 4
- Antigens which are preadsorbed onto an aluminium salt can be preadsorbed individually prior to mixing. In another embodiment, a mix of antigens may be preadsorbed prior to mixing with further adjuvants. In one embodiment, IPV may be adsorbed separately or as a mixture of IPV types 1 , 2 and 3.
- aluminium phosphate can be a precipitate of insoluble aluminium phosphate (amorphous, semi-crystalline or crystalline), which can be optionally but not exclusively prepared by mixing soluble aluminium salts and phosphoric acid salts.
- Alhydrogel aluminium hydroxide, 3% suspension in water
- Adjuphos aluminium phosphate, 2% suspension in saline
- Non-immunological components of vaccines of the invention are non-immunological components of vaccines of the invention.
- Vaccines of the invention will typically, in addition to the antigenic and adjuvant components mentioned above, comprise one or more "pharmaceutically acceptable carriers or excipients", which include any excipient that does not itself induce the production of antibodies harmful to the individual receiving the composition.
- Suitable excipients are typically large, slowly metabolised macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001 , Vaccine, 19:21 18), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes).
- Such carriers are well known to those of ordinary skill in the art.
- the vaccines may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, phosphate buffered physiologic saline is a typical carrier. A thorough discussion of pharmaceutically acceptable excipients is available in reference Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20 th edition, ISBN:0683306472.
- compositions of the invention may be lyophilised or in aqueous form, i.e. solutions or suspensions.
- Liquid formulations of this type allow the compositions to be administered direct from their packaged form, without the need for reconstitution in an aqueous medium, and are thus ideal for injection.
- Compositions may be presented in vials, or they may be presented in ready filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses (e.g. 2 doses).
- Liquid vaccines of the invention are also suitable for reconstituting other vaccines from a lyophilised form.
- the invention provides a kit, which may comprise two vials, or may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reconstitute the contents of the vial prior to injection.
- Vaccines of the invention may be packaged in unit dose form or in multiple dose form (e.g. 2 doses). For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition for injection has a volume of 0.5ml_.
- vaccines of the invention have a pH of between 6.0 and 8.0, in another embodiment vaccines of the invention have a pH of between 6.3 and 6.9, e.g. 6.6 ⁇ 0.2.
- Vaccines may be buffered at this pH. Stable pH may be maintained by the use of a buffer. If a composition comprises an aluminium hydroxide salt, a histidine buffer may be used (WO03/009869). The composition should be sterile and/or pyrogen free.
- Compositions of the invention may be isotonic with respect to humans.
- Vaccines of the invention may include an antimicrobial, particularly when packaged in a multiple dose format. Thiomersal should be avoided as this leads to loss of potency of the IPV component.
- antimicrobials such as 2- phenoxyethanol or parabens (methyl, ethyl, propyl parabens).
- Any preservative is preferably present at low levels.
- Preservative may be added exogenously and/or may be a component of the bulk antigens which are mixed to form the composition (e.g. present as a preservative in pertussis antigens).
- vaccines of the invention are thiomersal free or substantially thiomersal free.
- thiomersal free or substantially thiomersal free it is meant that there is not enough thiomersal present in the final formulation to negatively impact the potency of the IPV component. For instance, if thiomersal is used during the Pw or Hepatitis B surface antigen purification process it should be substantially removed prior to mixture with IPV.
- Thiomersal content in the final vaccine should be less than 0.025 ⁇ g/ ⁇ g protein,
- thiomersal is not added nor used in the purification of any component.
- Vaccines of the invention may comprise detergent e.g. a Tween (polysorbate), such as Tween 80.
- Detergents are generally present at low levels e.g. ⁇ 0.01 %.
- Vaccines of the invention may include sodium salts (e.g. sodium chloride) to give tonicity.
- the composition may comprise sodium chloride.
- the concentration of sodium chloride in the composition of the invention is in the range of 0.1 to 100 mg/mL (e.g. 1-50mg/ml_, 2-20mg/ml_, 5-15mg/ml_) and in a further embodiment the concentration of sodium chloride is 10 ⁇ 2mg/ml_ NaCI e.g. about 9mg/ml_.
- Vaccines of the invention will generally include a buffer.
- a phosphate or histidine buffer is typical.
- Vaccines of the invention may include free phosphate ions in solution (e.g. by the use of a phosphate buffer) in order to favour non-adsorption of antigens.
- the concentration of free phosphate ions in the composition of the invention is in one embodiment between 0.1 and 10.OmM, or in another embodiment between 1 and 5mM, or in a further embodiment about 2.5mM.
- the vaccines of the invention are formulated as a vaccine for in vivo administration to the host, such that they confer an antibody titre superior to the criterion for seroprotection for each antigenic component for an acceptable percentage of human subjects. This is an important test in the assessment of a vaccine's efficacy throughout the population. Antigens with an associated antibody titre above which a host is considered to be seroconverted against the antigen are well known, and such titres are published by organisations such as WHO.
- more than 80% of a statistically significant sample of subjects is seroconverted, in another embodiment more than 90% of a statistically significant sample of subjects is seroconverted, in a further embodiment more than 93% of a statistically significant sample of subjects is seroconverted and in yet another embodiment 96-100% of a statistically significant sample of subjects is seroconverted.
- the amount of antigen in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending on which specific immunogens are employed. Generally it is expected that each dose will comprise 1-1000 ⁇ g of total immunogen, or 1- 100 ⁇ g, or 1-40 ⁇ g, or 1-5 ⁇ g.
- a primary vaccination course may include 2-3 doses of vaccine, given one to two months apart, e.g. following the WHO recommendations for DTP immunisation.
- Vaccines of the invention can be packaged in various types of container e.g. in vials, in syringes, etc.
- a multidose vial will typically comprise a re-sealable plastic port through which a sterile needle can be inserted to remove a dose of vaccine, which reseals once the needle has been removed.
- the vaccine may be supplied in various containers (e.g. 2 or 3).
- the contents of the containers may be mixed extemporaneously before administering to a host in a single injection or it may be administered concomitantly at different sites.
- the dose of the vaccine will typically be 0.5ml_.
- kits comprising two multi-valent vaccines for conferring protection in a host against disease caused by poliovirus, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae and optionally one or more of Hepatitis B, Haemophilus influenza type B, Neisseria meningitidis type A, Neisseria meningitidis type C, Neisseria meningitidis type W, Neisseria meningitidis type Y, Neisseria meningitidis type B, Salmonella typhi, Hepatitis A or Malaria.
- the kit comprises a first container comprising:
- TT or T tetanus toxoid
- Pw killed whole-cell Bordetella pertussis
- Pa acellular pertussis components
- Hepatitis B surface antigen HepB or HB
- (2A) (a) conjugates of a carrier protein and a capsular saccharide N. meningitidis type A (MenA), N. meningitidis type C (MenC), N. meningitidis type W (Men W) and/or N. meningitidis type Y (Men Y) (see above for various Men saccharide combinations of the invention) [e.g. made by the conjugation process of the invention], and (b) optionally a conjugate of a carrier protein and the capsular saccharide of H. influenzae type B (Hib); or
- (2B) (a) a conjugate of a carrier protein and the capsular saccharide of H. influenzae type B (Hib), and
- the containers may in either case additionally comprise HepA antigen(s) and/or MenB antigen(s) and/or RTS, S and/or Streptococcus pneumonia antigen(s).
- the first container has in addition to components b), c), d) also a), e), f), g), e)+f), e)+g), f)+g) or e)+f)+g), a) + e), a) + f), a) + g), a) + e) + f), a) + e) + g), a) + f) + g), a) + e) + f) + g).
- the vaccine of the first container may be liquid and the vaccine of the second container may be either liquid or lyophilised (e.g. in the presence of a known stabilising excipient such as sucrose or trehalose).
- the containers of the kit can be packaged separately or, optionally, packed together.
- the kit is provided with a list of instructions for administration of the vaccines in the two or more containers.
- a container in a kit contains a certain saccharide conjugate
- the same conjugate is not present in the other containers of the kit.
- the vaccines of the first and second containers are administered concomitantly at different sites (as described below under "administration of vaccines of the invention), and in an alternative embodiment the inventors envision that the contents of the first and second containers may be mixed (optionally extemporaneously) before administration as a single vaccine.
- the present invention also provides a method for producing a vaccine formulation comprising the step of mixing the components of the vaccine together with a pharmaceutically acceptable excipient.
- a vaccine as herein described for use in a medicament for the treatment or prevention of diseases caused by infection by one or more of poliovirus, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Hepatitis B virus, Haemophilus influenzae, Neisseria meningitidis type A, Neisseria meningitidis type C, Neisseria meningitidis type W, Neisseria meningitidis type Y, Salmonella typhi or Hepatitis A.
- Clostridium tetani Corynebacterium diphtheriae, Hepatitis B virus, Haemophilus influenzae, Neisseria meningitidis type A, Neisseria meningitidis type C, Neisseria meningitidis type W, Neisseria meningitidis type Y, Salmonella typhi or Hepatitis A.
- each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented.
- each dose will comprise 0.1-100 ⁇ g of saccharide, in another embodiment each dose will comprise 0.1-50 ⁇ g, in a further embodiment each dose will comprise 0.1-10 ⁇ g, in yet another embodiment each dose will comprise 1 to 5 ⁇ g saccharide.
- the content of protein antigens in the vaccine will be in the range 1-100 ⁇ g, in another embodiment the content of the protein antigens in the vaccines will be in the range 5-50 ⁇ g, in a further embodiment the content of the protein antigens in the vaccines will be in the range 5 - 25 ⁇ g.
- Vaccine preparation is generally described in Vaccine Design ["The subunit and adjuvant approach” (eds Powell M. F. & Newman MJ.) (1995) Plenum Press New York].
- Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example by Likhite, US Patent 4,372,945 and by Armor et ai, US Patent 4,474,757.
- Use of Quil A is disclosed by Dalsgaard et al., 1977, Acta Vet Scand. 18:349.
- 3D-MPL is available from Ribi immunochem, USA and is disclosed in British Patent Application No. 222021 1 and US Patent 4,912,094.
- QS21 is disclosed in US Patent 5,057,540.
- the amount of saccharide conjugates per 0.5 ml. dose of bulk vaccine is less than 10 ⁇ g (of saccharide in the conjugate), in another embodiment the amount of conjugate is 1-7, in another embodiment the amount of conjugate is 2-6 ⁇ g, or in a further embodiment about 2.5, 3, 4 or 5 ⁇ g.
- certain components for example DTPw components, can be combined separately before adding the adsorbed HBsAg or other components.
- the combined vaccine compositions according to any aspect of the invention can be prepared as follows: The IPV, DTPw, HepB, MenA, MenC, MenW, MenY, MenB, Vi, Hepatitis A or other components are pre-adsorbed onto a suitable adjuvant, especially aluminium hydroxide or aluminium phosphate or a mixture of both. After allowing time for complete and stable adsorption of the respective components, the different components are combined under appropriate conditions.
- the Hib, Vi, MenA, MenC, MenW and/or MenY conjugate(s) may or may not be adsorbed onto aluminium adjuvant salt before being mixed with the DTPw vaccine.
- vaccines of the invention are prepared at between 15°C and
- 30 0 C (e.g. between 19°C and 27°C, or at 23 ⁇ 4°C).
- the invention provides a method for raising an immune response in a mammal, comprising the step of administering an effective amount of a vaccine of the invention.
- the vaccines can be administered prophylactically (i.e. to prevent infection) or therapeutically (i.e. to treat disease after infection).
- the immune response is preferably protective and preferably involves antibodies.
- the method may raise a booster response.
- Dosing treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule.
- a primary dose schedule which may be in the first year of life, may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting can be routinely determined.
- the mammal is a human.
- the human is preferably a child (e.g. a toddler of infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably an adult.
- a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
- the vaccine preparations of the present invention may be used to protect or treat a mammal susceptible to infection, by means of administering said vaccine directly to a patient.
- Direct delivery may be accomplished by parenteral injection (intramuscularly, intraperitoneal ⁇ , intradermally, subcutaneously, intravenously, or to the interstitial space of a tissue); or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration.
- administration is by intramuscular injection to the thigh or the upper arm. Injection may be via a needle (e.g. a hypodermic needle), but needle free injection may alternatively be used.
- a typical intramuscular dose is 0.5ml_.
- compositions of the invention may be prepared in various forms.
- the compositions may be prepared as injectables, either as liquid solutions or suspensions.
- the composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or spray.
- the composition may be prepared as a suppository or pessary.
- the composition may be prepared for nasal, aural or ocular administration e.g. as spray, drops, gel or powder (see e.g. Almeida & Alpar, 1996, J Drug Targeting, 3:455; Bergquist et al., 1998, APMIS, 106:800).
- Successful intranasal administration of DTP vaccines has been reported (Ryan et al., 1999, Infect. Immun., 67:6270; Nagai et al., 2001 , Vaccine, 19:4824).
- the vaccines of the first and second (and third where applicable) containers are administered concomitantly at different sites, and in an alternative embodiment the inventors envision that the contents of the first and second containers may be mixed (optionally extemporaneously) before administration as a single vaccine.
- the invention may be used to elicit systemic and/or mucosal immunity.
- One way of checking the efficacy of therapeutic treatment involves monitoring bacterial infection after administration of the composition of the invention.
- One way of checking efficacy of prophylactic treatment involves monitoring immune responses against the antigens after administration of the composition, lmmunogenicity of compositions of the invention can be determined by administering them to test subjects (e.g. children 12-16 months age, or animal models - WO 01/30390) and then determining standard immunological parameters. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. Rather than assessing actual protective efficacy in patients, standard animal and in vitro models and correlates of protection for assessing the efficacy of DTP vaccines are well known.
- Example 1 a - preparation of meningococcal MenA and MenC capsular polysaccharide conjugate according to the invention
- MenC -TT conjugates were produced using native polysaccharides (of over 15OkDa as measured by MALLS) or were slightly microfluidised. MenA-TT conjugates were produced using either native polysaccharide or slightly microfluidised polysaccharide of over 6OkDa as measured by the MALLS method of example 2. Sizing was by microfluidisation using a homogenizer Emulsiflex C-50 apparatus. The polysaccharides were then filtered through a 0.2 ⁇ m filter.
- MenA capsular polysaccharide to tetanus toxoid via a spacer
- the covalent binding of the polysaccharide and the spacer (ADH) is carried out by a coupling chemistry by which the polysaccharide is activated under controlled conditions by a cyanylating agent, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP).
- CDAP 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate
- the spacer reacts with the cyanylated PS through its hydrazino groups, to form a stable isourea link between the spacer and the polysaccharide.
- the PSA AH solution was concentrated to a quarter of its initial volume and then diafiltered with 30 volumes of 0.2M NaCI using a Filtron Omega membrane with a cut-off of 1OkDa, and the retentate was filtered.
- the purified TT solution and the PSA AH solution were diluted to reach a concentration of 10 mg/ml for PSA AH and
- EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) was added to the PS AH solution (2g saccharide) in order to reach a final ratio of 0.9 mg EDAC/mg PSA AH .
- the pH was adjusted to 5.0.
- the purified tetanus toxoid was added with a peristaltic pump (in 60 minutes) to reach 2 mg TT/mg PSA AH .
- the resulting solution was left 60 min at +25°C under stirring to obtain a final coupling time of 120 min.
- the solution was neutralised by addition of 1 M Tris-Hcl pH 7.5 (1/10 of the final volume) and left 30 minutes at +25°C then overnight at +2°C to +8°C.
- the conjugate was clarified using a 10 ⁇ m filter and was purified using a Sephacryl S400HR column (Pharmacia, Sweden). The column was equilibrated in 10 mM Tris-HCI (pH 7.0), 0.075 M NaCI and the conjugate (approx. 66OmL) was loaded on the column (+2°C to +8°C). The elution pool was selected as a function of optical density at 280 nm. Collection started when absorbance increased to 0.05. Harvest continued until the Kd reached 0.30. The conjugate was filter sterilised at +20 0 C, then stored at +2°C to +8°C. The resultant conjugate had a polysaccharide:protein ratio of 1 :2-1 :4 (w/w).
- MenC capsular polysaccharide to tetanus toxoid via a spacer
- the covalent binding of the polysaccharide and the spacer (ADH) is carried out by a coupling chemistry by which the polysaccharide is activated under controlled conditions by a cyanylating agent, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP).
- CDAP 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate
- the spacer reacts with the cyanylated PS through its hydrazino groups, to form a stable isourea link between the spacer and the polysaccharide.
- a 20mg/ml solution of MenC (pH6.0) (3.5 g) was treated with a freshly prepared 100mg/ml solution of CDAP in acetonitrile/water (50/50 (v/v)) to obtain a CDAP/MenC ratio of 1.5 (w/w).
- the pH was raised to pH 10.0.
- pH 5M NaCI was added to achieve a final concentration of 2M NaCI.
- ADH was added to obtain an ADH/MenC ratio of 8.9.
- the pH of the solution was decreased to 8.75 and the reaction proceeded for 2 hours (retained at 25 0 C).
- the PSC AH solution was concentrated to a minimum of 150 ml. and then diafiltered with 30 volumes of 0.2M NaCI using a Filtron Omega membrane with a cut-off of 1 OkDa, and the retentate was filtered.
- the purified TT solution and the PSC AH solution (2g scale) were diluted in 0.2M NaCI to reach a concentration of 15 mg/ml for PSC AH and 20mg/ml for TT.
- the purified tetanus toxoid was added to the PSC AH solution in order to reach 2 mg TT/mg PSC AH -
- the pH was adjusted to 5.0.
- EDAC (16.7 mg/ml in Tris 0.1 M pH 7.5) was added with a peristaltic pump (in 10 minutes) to reach a final ratio of 0.5 mg EDAC/mg PSC AH -
- the resulting solution was left 1 10 min at +25°C under stirring and pH regulation to obtain a final coupling time of 120 min.
- the solution was then neutralized by addition of 1 M Tris- HcI pH 9.0 (1/10 of final volume) and left 30 minutes at +25°C then overnight at +2°C to +8°C.
- the conjugate was clarified using a 10 ⁇ m filter and was purified using a Sephacryl S400HR column (Pharmacia, Sweden). The column was equilibrated in 10 mM Tris-HCI (pH 7.0), 0.075 M NaCI and the conjugate (approx. 46OmL) was loaded on the column (+2°C to +8°C). The elution pool was selected as a function of optical density at 280 nm. Collection started when absorbance increased to 0.05. Harvest continued until the Kd reached 0.20. The conjugate was filter sterilised at +20 0 C, then stored at +2°C to +8°C.
- the resultant conjugate had a polysaccharide:protein ratio of 1 :2-1 :4 (w/w).
- Various experiments adding EDAC over 10-45 minutes were carried out - in each case good quality MenC conjugates resulted. If, however the TT carrier was added last slowly to the MenC-ADH + EDAC mix this led to a gel - a conjugate that could not be purified.
- MenCAH after CDAP treatment with ADH about 3.47% of hydroxyl groups were derivatized with ADH (with an estimation of two available hydroxyl groups per repeat subunit).
- MenA about 1 1.5% of hydroxyl groups derivatized with ADH (considering there is only one available hydroxyl group per repeat unit).
- PS03-TT ⁇ H process PS03-TT ⁇ H 208
- PS was weighed on the basis of 10% theoretical moisture content.
- the native PS was dissolved overnight in 2M NaCI at an initial concentration of 3 mg/ml. Before sizing, the solution of native PS was clarified on 5 ⁇ m cut-off filter.
- a homogenizer EMULSIFLEX C-50 apparatus was used to reduce the molecular weight and the viscosity of the polysaccharide before the activation step.
- the efficiency of the sizing depends on the circuit pressure, the plunger alimentation pressure and on the total cycles number.
- the homogenizing cell of Emulsiflex was replaced with a cell with a fixed geometry (Micro fluidics F20Y-0.75 ⁇ m interaction chamber). The aim of the sizing was to reduce the molecular weight and the viscosity of the PS without a critical decrease of its antigenicity.
- the size reduction was done at 6000 ⁇ 500 psi and followed in-process by a measure of viscosity. The sizing was stopped when the target of 2.0 ⁇ 0.2 cp was reached.
- the derivatization step was performed at 25°C under continuous stirring in a T° controlled waterbath.
- TT was diluted in NaCI 0.2M to obtain a final TT concentration of 25mg/ml.
- ADH was added in solid form to the TT solution to reach a 0.2M final concentration.
- the solution was set at pH 6.2+/- 0.1 with HCI.
- EDAC was then added to the TT/ADH solution to reach a final 0.02M concentration.
- the pH was set at 6.2+/-0.1 with HCI and was kept under pH regulation during 1 hour.
- the pH was raised up to pH9.5 with NaOH to stop the reaction. The solution was left during 2 hours under pH regulation before the diafiltration step.
- TT AH derivative was diafiltered in order to remove unreacted ADH and EDAC by-products.
- the diafiltration was performed on a centramate membrane (0.09 m 2 , 10 kDa cut-off).
- the solution was dialysed against 20 volumes of 0.2M NaCI.
- the follow-up of the diafiltration step was performed by a quantification of ADH (TNBS assay) in the permeate after 5, 10, 15 and 20 volumes of diafiltration.
- TT AH was finally filtered on 0.22 ⁇ m cut-off membrane (Millipack 40) at a flow-rate of 10 ml/min.
- the filtered TT AH was then stored at -70 0 C
- PS3 50mg of PS3 were diluted in 2M NaCI to obtain a final PS concentration of 2 mg/ml.
- the purified TT AH solution was diluted in 0.2M NaCI to reach a concentration of 10 mg/ml.
- the PS3 solution was adjusted to pH5 with HCI.
- EDAC was added in solid form to the PS3 solution in order to reach a final concentration of 0.5 mg EDAC/ mg PS.
- the pH was adjusted to 5.0 ⁇ 0.05 with HCI and TT AH was manually added in 1 1 minutes (aliquots/min).
- the resulting solution was incubated 109 min at + 25°C with stirring and pH regulation to obtain a final coupling time of 120 min.
- the solution was neutralized by addition of 1 M Tris-HCI pH 7.5 and left 30 min at +25°C.
- the conjugate was finally clarified on a 5 ⁇ m membrane and injected on a Sephacryl
- PS03-TTAH process PS03AH-TT215
- the derivatization step was performed at 25°C under continuous stirring in a T° controlled waterbath.
- PS3 was diluted in NaCI 2M to obtain a final PS concentration of 3mg/ml.
- the PS solution was set at pH6.0 before the addition of CDAP (0.25mg/mg PS, dissolution at 100mg/ml in a mix of acetonitrile/ WFI).
- the pH was increased to pH9.5 with NaOH before the addition of ADH (8.9mg ADH/mg PS, dissolution at 100mg/ml in 0.2M NaCI).
- the pH was kept at 9.5 and regulated during 60 minutes.
- the percentage of derivatization corresponded to 2.4% (2.4 mg ADH/ 100 mg PS).
- TNBS for the estimating ADH
- DMAB or resorcinol Monsigny et al (1988) Anal. Biochem. 175, 525-530
- PS dosage was 228 ⁇ g/ml
- PS dosage 5250 ⁇ g/ml
- Mw of ADH 174.2
- Mw of the repeat unit of PS3 338.27 (having 1 COOH and 4 OH groups)
- PS3 AH derivative was diafiltered in order to remove unreacted ADH and CDAP byproducts.
- the diafiltration was performed on a UFP-30-C-H24LA membrane (42 cm 2 , 30 kDa cut-off).
- the solution was dialysed against 20 volumes of 0.2M NaCI.
- the follow-up of the diafiltration step was performed by a quantification of ADH (TNBS assay) in the permeate after 5, 10, 15 and 20 volumes of diafiltration.
- PS AH was finally filtered on 0.22 ⁇ m cut-off membrane (Millipack 40) at a flow-rate of 10 ml/min.
- the filtered PS3 AH was then stored at 4°C.
- PS3 AH 50mg of PS3 AH was diluted in 0.2M NaCI to obtain a final PS concentration of 2 mg/ml.
- the purified TT solution was diluted in 0.2M NaCI to reach a concentration of 10 mg/ml.
- the PS3 AH solution was adjusted to pH5 with HCI.
- EDAC was added in solid form to the PS3 AH solution in order to reach a final concentration of 0.5 mg EDAC/ mg PS.
- the pH was adjusted to 5.0 ⁇ 0.05 with HCI and TT was added in 10 minutes using a peristaltic pump.
- the resulting solution was incubated 1 10 min at + 25°C with stirring and pH regulation to obtain a final coupling time of 120 min.
- the solution was neutralized by addition of 1 M Tris-HCI pH 7.5 and left 30 min at +25°C.
- the conjugate was finally clarified on a 5 ⁇ m membrane and injected on a Sephacryl S400HR column.
- PS3 AH 50mg of PS3 AH was diluted in 0.2M NaCI to obtain a final PS concentration of 2 mg/ml.
- the purified TT solution was diluted in 0.2M NaCI to reach a concentration of 10 mg/ml.
- the PS3 AH and TT solutions were mixed and adjusted to pH5 with HCI.
- EDAC was dissolved in a Tris 1 M pH7.5 buffer. 40 ⁇ l of EDAC were added each minute
- the derivatization step was performed at 25°C with continuous stirring in a T° controlled waterbath.
- PS3 was diluted in NaCI 2M to obtain a final PS concentration of 3mg/ml.
- EDAC was added in solid form to reach an EDAC/ PS ratio of 0.1 mg/mg PS.
- the pH of the solution was set at 5.
- ADH (8.9mg ADH/mg PS, dissolution at 100mg/ml in 0.2M NaCI) was then added using a peristaltic pump in 44 minutes (though as such an excess of ADH was present, direct addition would also have been OK).
- the pH was kept at 5.0+/-0.1 and regulated during 120 minutes (44' + 76').
- the reaction was stopped by increasing the pH to 7.5 using sodium hydroxide.
- the percentage of derivatization corresponded to 3.7% (mg ADH/ mg PS).
- PS3 carboxyl groups were ADH modified COOH groups.
- PS3 AH derivative was diafiltered in order to remove unreacted ADH and EDAC byproducts.
- the diafiltration was performed on a UFP-30-C-H24LA membrane (42 cm 2 , 30 kDa cut-off).
- the solution was dialysed against 23 volumes of 0.2M NaCI.
- the follow-up of the diafiltration step was performed by a quantification of ADH (TNBS assay) in the permeate after 5, 10, 15 and 20 volumes of diafiltration
- PS AH was finally filtered on 0.22 ⁇ m cut-off membrane (Millipack 40) at a flow-rate of 10 ml/min.
- the filtered PS3 AH was then stored at 4°C.
- PS3 AH 50mg of PS3 AH was diluted in 0.2M NaCI to obtain a final PS concentration of 2 mg/ml.
- the purified TT solution was diluted in 0.2M NaCI to reach a concentration of 10 mg/ml.
- the PS3 AH and TT solutions were mixed together.
- the pH was adjusted to 5.0 ⁇ 0.05 with HCI and EDAC was manually added in 10 minutes (equal part-aliquots added each minute).
- the resulting solution was incubated 1 10 min at + 25°C with stirring and pH regulation to obtain a final coupling time of 120 min.
- the conjugate was finally clarified on a 5 ⁇ m membrane and injected on a Sephacryl S400HR column.
- the last component added in the reaction solution can be either the TT protein or the EDAC reagent.
- the time of addition can have an effect on the resulting conjugates.
- EDAC is added first to the PS AH (having both reactive amino and carboxyl groups) this can lead to intra cross-linking of hydrazine and carboxylic groups present on the polysaccharide, and thus could lead to a more cross-linked conjugate with a weaker final ratio after the addition of TT in 10 minutes.
- Example 1 c - preparation of S. typhi Vi polysaccharide conjugate of the invention
- PS was weighed on the basis of 15% theoretical moisture content.
- the native PS is dissolved overnight in WFI at an initial concentration of 7 mg/ml.
- the solution of native PS is clarified on 10 ⁇ m cut-off filter at a flow-rate of 50 ml/min.
- a homogenizer EMULSIFLEX C-50 apparatus was used to reduce the molecular weight and the viscosity of the polysaccharide before the activation step. The efficiency of the sizing depends on the circuit pressure, the plunger alimentation pressure and on the total cycles number.
- the homogenizing cell of Emulsiflex was replaced by a cell with a fixed geometry (Microfluidics F20Y-0.75 ⁇ m interaction chamber).
- the aim of the sizing is to reduce the molecular weight and the viscosity of the PS without a critical decrease of its antigenicity.
- the size reduction was realized at 15000 ⁇ 500 psi and followed in-process by a measure of viscosity. The sizing is stopped when the target of 5.0 ⁇ 0.3 cp is reached.
- Sized PS is filtered on a Millipak 40 membrane (cut-off 0.22 mm) at a flow-rate of 10 ml/min.
- the filtered sized PS is stored at -20 0 C.
- TNBS dosage was 200 ⁇ g/ml and PS dosage was 4034 ⁇ g/ml; thus 0.0697 ⁇ moles of ADH / 16.46 ⁇ mole of repeat unit (Mw 245). 1.3 ⁇ moles of ADH / 16.46 ⁇ mole of reactive COOH group on Vi, thus 7% of Vi COOH groups were ADH modified COOH groups.
- PSVi AH derivative was diafiltered in order to remove unreacted ADH and EDAC byproducts.
- the diafiltration was performed on a centramate membrane (0.09 m 2 , 10 kDa cut-off).
- the solution was dialysed against 20 volumes of 0.2M NaCI.
- the follow-up of the diafiltration step was performed by a quantification of ADH (TNBS assay) in the permeate after 3, 5, 10 and 20 volumes of diafiltration
- PSVi AH was finally filtered on 0.22 ⁇ m cut-off membrane (Millipack 40) at a flow-rate of 10 ml/min.
- the filtered PSViAH was stored at +2/+8°C for a maximum of 4 days.
- TT was added to the PSVi AH solution in order to reach a final ratio of 2.5 mg TT/ mg PS.
- the pH is adjusted to 5.0 ⁇ 0.05 with 1 N HCI.
- the EDAC solution (7.5 mg/ml in 0.1 M Tris pH 7.5) was then added (in 10 minutes with a peristaltic pump) to reach 0.25 mg EDAC/ mg PSVi AH -
- the resulting solution was incubated 50 min at + 25°C with stirring and pH regulation to obtain a final coupling time of 60 min.
- the solution was neutralized by addition of 1 M Tris-HCI pH 7.5 and left 30 min at +25°C.
- the conjugate was transferred at 4°C and is left overnight under continuous slow stirring before the chromatography step. Purification
- the bulk was brought back to room temperature. Then the conjugate was filtered on an Opticap 4" sterilizing membrane. The flow rate was fixed at 30 ml/min.
- the resulting conjugate had a final TT/PS ratio (w/w) of 2.44/1, a free PS content of 3.7% and a ⁇ PS/ ⁇ PS antigenicity of 58%.
- Example 1 c(ii) - other means to prepare of S. typhi Vi polysaccharide conjugates of the invention
- Example 1c is carried out, but the following conditions are altered:
- Hib PRP polysaccharide was activated by adding CNBr and incubating at pH10.5 for 6 minutes. The pH was lowered to pH8.75 and adipic acid dihydrazide (ADH) was added and incubation continued for a further 90 minutes. The activated PRP was coupled to purifed tetanus toxoid via carbodiimide condensation using 1-ethyl-3-(3- dimethyl-aminopropyl)carbodiimide (EDAC).
- EDAC 1-ethyl-3-(3- dimethyl-aminopropyl)carbodiimide
- EDAC was added to the activated PRP to reach a final ratio of 0.6mg EDAC/mg activated PRP.
- the pH was adjusted to 5.0 and purified tetanus toxoid was added to reach 2mg TT/mg activated PRP.
- the resulting solution was left for three days with mild stirring. After filtration through a 0.45 ⁇ m membrane, the conjugate was purifed on a sephacryl S500HR (Pharmacia, Sweden) column equilibrated in 0.2M NaCI.
- MenC -TT conjugates were produced using native polysaccharides (of over 15OkDa as measured by MALLS) or were slightly microfluidised. MenA-TT conjugates were produced using either native polysaccharide or slightly microfluidised polysaccharide of over 6OkDa as measured by the MALLS method of example 2. MenW and MenY-TT conjugates were produced using sized polysaccharides of around 100-20OkDa as measured by MALLS (see example 2). Sizing was by microfluidisation using a homogenizer Emulsiflex C-50 apparatus. The polysaccharides were then filtered through a 0.2 ⁇ m filter.
- the polysaccharide at a concentration of 10-20mg/ml in 2M NaCI pH 5.5-6.0 was mixed with CDAPsolution (100mg/ml freshly prepared in acetonitrile/WFI, 50/50) to a final CDAP/polysaccharide ratio of 0.75/1 or 1.5/1.
- CDAPsolution 100mg/ml freshly prepared in acetonitrile/WFI, 50/50
- the pH was raised with sodium hydroxide to pH10.0.
- tetanus toxoid was added to reach a protein/polysaccharide ratio of 1.5/1 for MenW, 1.2/1 for MenY, 1.5/1 for MenA or 1.5/1 for MenC.
- glycine was added to a final ratio of glycine/PS (w/w) of 7.5/1 and the pH was adjusted to pH9.0. The mixture was left for 30 minutes.
- the conjugate was clarified using a 10 ⁇ m Kleenpak filter and was then loaded onto a Sephacryl S400HR column using an elution buffer of 15OmM NaCI, 1OmM or 5mM Tris pH7.5. Clinical lots were filtered on an Opticap 4 sterilizing membrane.
- the resultant conjugates had an average polysaccharide:protein ratio of 1 :1-1 :5 (w/w).
- Detectors were coupled to a HPLC size exclusion column from which the samples were eluted.
- the laser light scattering detector measured the light intensities scattered at 16 angles by the macromolecular solution and on the other hand, an interferometric refractometer placed on-line allowed the determination of the quantity of sample eluted. From these intensities, the size and shape of the macromolecules in solution can be determined.
- the mean molecular weight in weight (M w ) is defined as the sum of the weights of all the species multiplied by their respective molecular weight and divided by the sum of weights of all the species.
- Root mean square radius: -Rw- and R 2 W is the square radius defined by:
- the polydispersity is defined as the ratio -Mw / Mn-.
- Meningococcal polysaccharides were analysed by MALLS by loading onto two HPLC columns (TSKG6000 and 5000PWxI) used in combination. 25 ⁇ l of the polysaccharide were loaded onto the column and was eluted with 0.75ml of filtered water.
- the polyaccharides are detected using a light scattering detector ( Wyatt Dawn DSP equipped with a 1 OmW argon laser at 488nm) and an inferometric refractometer ( Wyatt Otilab DSP equipped with a P100 cell and a red filter at 498nm).
- the molecular weight polydispersities and recoveries of all samples were calculated by the Debye method using a polynomial fit order of 1 in the Astra 4.72 software.
- the blood samples were used to asess the percentage of SBA-MenA, SBA-MenC, SBA- MenW135 and SBA-MenY responders one month after the vaccine dose.
- a vaccine response was defined as 1 ) for initially seronegative subjects - a post-vaccination antibody titre > 1/32 at 1 month or 2) for initially seropositive subjects - antibody titre of > 4 fold the pre-vaccination antibody titre.
- the use of a spacer in the MenA conjugate led to an increased immune response against MenA.
- the percentage of responders rose from 66% to 90-95% when the AH spacer was added. This was reflected in an increase in SBA GMT from 4335 to 10000 and an increase in GMC from 5 to 20-40.
- the use of a AH spacer also led to an increased immune response against MenC as seen by an increase in the percentage of responders and an increase in the SBA GMT.
- An increase could also be seen in the SBA-GMT against MenY (6742-7122) and against MenW (4621-5418) when a spacer was introduced.
- Example 4 Clinical trial assessing the effect of a linker in MenA and MenC conjugates in a MenACWY conjugate vaccine
- MenACWY vaccine A single dose of different formulations of MenACWY vaccine was administered to teenagers of 15-19 years in 5 groups of 25 subjects in a 1 :1 :1 :1 :1 randomised trial.
- the formulations tested were: F1 - MenACWY conjugated to tetanus toxoid with the MenA and MenC conjugates containing an AH spacer (made according to example 1 ) - 2.5/2.5/2.5/2.5 ⁇ g
- the blood samples were used to asess the percentage of SBA-MenA, SBA-MenC, SBA- MenW135 and SBA-MenY responders one month after the vaccine dose.
- a vaccine response was defined as 1 ) for initially seronegative subjects - a post-vaccination antibody titre > 1/32 at 1 month or 2) for initially seropositive subjects - antibody titre of > 4 fold the pre-vaccination antibody titre.
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CN103083657B (zh) * | 2005-06-27 | 2016-06-08 | 葛兰素史密丝克莱恩生物有限公司 | 免疫原性组合物 |
BRPI0818545A2 (pt) * | 2007-10-19 | 2017-07-04 | Novartis Ag | formulações de vacinais meningocócicas |
GB0810894D0 (en) * | 2008-06-13 | 2008-07-23 | Novartis Vaccines & Diagnostic | Conjugated saccharide |
GB0822633D0 (en) | 2008-12-11 | 2009-01-21 | Novartis Ag | Formulation |
GB0822634D0 (en) * | 2008-12-11 | 2009-01-21 | Novartis Ag | Meningitis vaccines |
EP3199177A1 (en) * | 2009-10-30 | 2017-08-02 | GlaxoSmithKline Biologicals S.A. | Purification of staphylococcus aureus type 5 and type 8 capsular saccharides |
KR102447303B1 (ko) | 2013-08-24 | 2022-09-23 | 브하라트 바이오테크 인터내셔날 리미티드 | 박테리아성 백신 및 그의 제조방법 |
EP2870974A1 (en) * | 2013-11-08 | 2015-05-13 | Novartis AG | Salmonella conjugate vaccines |
US9815886B2 (en) | 2014-10-28 | 2017-11-14 | Adma Biologics, Inc. | Compositions and methods for the treatment of immunodeficiency |
FI3506935T3 (fi) * | 2016-09-02 | 2024-04-17 | Sanofi Pasteur Inc | Neisseria meningitidis -rokote |
US10259865B2 (en) | 2017-03-15 | 2019-04-16 | Adma Biologics, Inc. | Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection |
JOP20200214A1 (ar) * | 2019-09-03 | 2021-03-03 | Serum Institute Of India Pvt Ltd | تركيبات مولدة للمناعة ضد الأمراض المعوية وطرق لتحضيرها |
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WO1998026799A1 (en) * | 1996-12-18 | 1998-06-25 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Conjugate vaccine for salmonella paratyphi a |
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US5180815A (en) * | 1988-04-13 | 1993-01-19 | Fuji Photo Film Co., Ltd. | Modified protein for carrying hapten |
US5965714A (en) * | 1997-10-02 | 1999-10-12 | Connaught Laboratories, Inc. | Method for the covalent attachment of polysaccharides to protein molecules |
EP1073667A2 (en) * | 1998-04-28 | 2001-02-07 | Galenica Pharmaceuticals, Inc. | Polysaccharide-antigen conjugates |
SI1549338T1 (sl) * | 2002-10-11 | 2011-04-29 | Novartis Vaccines & Diagnostic | Polipeptidna cepiva za ĺ iroko zaĺ äśito pred hipervirulentnimi miningokoknimi rodovi |
GB0405787D0 (en) * | 2004-03-15 | 2004-04-21 | Chiron Srl | Low dose vaccines |
GB0500787D0 (en) * | 2005-01-14 | 2005-02-23 | Chiron Srl | Integration of meningococcal conjugate vaccination |
GB0505518D0 (en) * | 2005-03-17 | 2005-04-27 | Chiron Srl | Combination vaccines with whole cell pertussis antigen |
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HALSEY N A, CLINICAL INFECTIOUS DISEASES 2001,33:S312 - S318 * |
JUN WANG ET AL: "An active immunization approach to generate protective catalytic antibodies", BIOCHEMICAL JOURNAL, vol. 360, no. 1, 15 November 2001 (2001-11-15), pages 151 - 157, XP055063555, ISSN: 0264-6021, DOI: 10.1042/0264-6021:3600151 * |
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